This application claims priority under 35 U.S.C. 119 of Danish application PA 2000 00045 filed Jan. 12, 2000, the contents of which are fully incorporated herein by reference.
The present invention relates to variants of pullulanases and to methods for constructing such variants.
Starches such as corn, potato, wheat, manioc and rice starch are used as the starting material in commercial large scale production of sugars, such as high fructose syrup, high maltose syrup, maltodextrins, amylose, G4-G6 oligosaccharides and other carbohydrate products such as fat replacers.
Degradation of Starch
Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains xcex1-1,4 D-glucose residues are joined by xcex1-1,6 glucosidic linkages. Amylopectin is partially degraded by xcex1-amylase, which hydrolyzes the 1,4-xcex1-glucosidic linkages to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin by xcex1-amylase results in the formation of so-called xcex1-limit dextrins which are not susceptible to further hydrolysis by the xcex1-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can be depolymerized to D-glucose by glucoamylase, which hydrolyzes linear oligosaccharides into D-glucose.
Amylose is a linear polysaccharide built up of D-glucopyranose units linked together by xcex1-1,4 glucosidic linkages. Amylose is degraded into shorter linear oligosaccharides by xcex1-amylase, the linear oligosaccharides being depolymerized into D-glucose by glucoamylase.
In the case of converting starch into a sugar, the starch is depolymerized. The depolymerization process consists of a pretreatment step and two or three consecutive process steps, namely a liquefaction process, a saccharification process and, depending on the desired end product, optionally an isomerization process.
Pre-treatment of Native Starch
Native starch consists of microscopic granules which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this xe2x80x9cgelatinizationxe2x80x9d process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or xe2x80x9cliquefiedxe2x80x9d so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation.
Liquefaction
During the liquefaction step, the long-chained starch is degraded into smaller branched and linear units (maltodextrins) by an xcex1-amylase (e.g. Termamyl(trademark), available from Novo Nordisk A/S, Denmark). The liquefaction process is typically carried out at about 105-110xc2x0 C. for about 5 to 10 minutes followed by about 1-2 hours at about 95xc2x0 C. The pH generally lies between about 5.5 and 6.2. In order to ensure an optimal enzyme stability under these conditions, calcium is added, e.g. 1 mM of calcium (40 ppm free calcium ions). After this treatment the liquefied starch will have a xe2x80x9cdextrose equivalentxe2x80x9d (DE) of 10-15.
Saccharification
After the liquefaction process the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g. AMG(trademark), available from Novo Nordisk A/S) and a debranching enzyme, such as an isoamylase (see e.g. U.S. Pat. No. 4,335,208) or a pullulanase (e.g. Promozyme(copyright), available from Novo Nordisk A/S; see U.S. Pat. No. 4,560,651). Before this step the pH is reduced to a value below 4.5, e.g. about 3.8, maintaining the high temperature (above 95xc2x0 C.) for a period of e.g. about 30 min. to inactivate the liquefying xcex1-amylase to reduce the formation of short oligosaccharides called xe2x80x9cpanose precursorsxe2x80x9d which cannot be hydrolyzed properly by the debranching enzyme.
The temperature is then lowered to 60xc2x0 C., glucoamylase and debranching enzyme are added, and the saccharification process proceeds for about 24-72 hours.
Normally, when denaturing the xcex1-amylase after the liquefaction step, a small amount of the product comprises panose precursurs which cannot be degraded by pullulanases or AMG. If active amylase from the liquefaction step is present during saccharification (i.e. no denaturing), this level can be as high as 1-2% or even higher, which is highly undesirable as it lowers the saccharification yield significantly. For this reason, it is also preferred that the xcex1-amylase is one which is capable of degrading the starch molecules into long, branched oligosaccharides (such as, e.g., the Fungamyl(trademark)-like xcex1-amylases) rather than shorter branched oligosaccharides.
Isomerization
When the desired final sugar product is e.g. high fructose syrup, the dextrose syrup may be converted into fructose by enzymatic isomerization. After the saccharification process the pH is increased to a value in the range of 6-8, preferably about pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase (such as Sweetzyme(trademark), available from Novo Nordisk A/S).
Debranching Enzymes
Debranching enzymes which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41), respectively. Isoamylase hydrolyses xcex1-1,6-D-glucosidic branch linkages in amylopectin and xcex2-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by their limited action on xcex1-limit dextrins.
When an acidic stabilized xcex1-amylase is used for the purpose of maintaining the amylase activity during the entire saccharification process (no inactivation), the degradation specificity should be taken into consideration. It is desirable in this regard to maintain the xcex1-amylase activity throughout the saccharification process, since this allows a reduction in the amyloglucidase addition, which is economically beneficial and reduces the AMG(trademark) condensation product isomaltose, thereby increasing the DE (dextrose equivalent) yield.
It will be apparent from the above discussion that the known starch conversion processes are performed in a series of steps, due to the different requirements of the various enzymes in terms of e.g. temperature and pH. It would therefore be desirable to be able to engineer one or more of these enzymes, e.g. pullulanases, so that the overall process could be performed in a more economical and efficient manner. One possibility in this regard is to engineer the otherwise thermolabile pullulanases so as to render them more stable at higher temperatures.
The inventors have modified the amino acid sequence of a pullulanase to obtain variants with improved properties, based on the three-dimensional structure of the pullulanase Promozyme(copyright) (available from Novo Nordisk A/S). The variants have altered physicochemical properties, e.g. an altered pH optimum, improved thermostability, increased specific activity or an altered cleavage pattern.
Accordingly, the object of the present invention is to provide a method for constructing pullulanases having altered properties, in particular to provide a method for constructing pullulanases having improved thermostability, altered pH dependent activity and/or altered substrate specificity, such as increased isoamylase activity.
Thus, in its broadest aspect, the present invention relates to a method for constructing a variant of a parent pullulanase, wherein the variant has at least one altered property as compared to said parent pullulanase, which method comprises:
i) analyzing the structure of the pullulanase to identify, on the basis of an evaluation of structural considerations, at least one amino acid residue or at least one structural region of the pullulanase, which is of relevance for altering said property;
ii) constructing a variant of the pullulanase, which as compared to the parent pullulanase, has been modified in the amino acid residue or structural part identified in i) so as to alter said property; and
iii) testing the resulting pullulanase variant for said property.
The property which may be altered by the above methods of the present invention may be, e.g., thermostability, pH dependent activity, specific activity, or substrate specificity. Thus, the variant may have, e.g., increased thermostability, higher activity at a lower pH, an altered pH optimum, improved thermostability, or increased specific activity, such as increased isoamylase activity.
Although it has been described in the following that modification of the parent pullulanase in certain regions and/or positions is expected to confer a particular effect to the thus produced pullulanase variant (such as an improved thermostability or an increased isoamylase activity), it should be noted that modification of the parent pullulanase in any of such regions may also give rise to any other of the above-mentioned effects. For example, any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability, may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased isoamylase activity.
Further aspects of the present invention relates to variants of a pullulanase, the DNA encoding such variants and methods of preparing the variants. Still further aspects of the present invention relates to the use of the variants for various industrial purposes, in particular for processes where sweeteners are made from starch. Other aspects of the present invention will be apparent from the below description as well as from the appended claims.